Human cytomegalovirus (HCMV) is an opportunistic pathogen with the ability to establish and maintain a lifelong relationship with its host, a persistent state termed latency. Primary infections and reactivation of latent HCMV results in a high viral load in patients with inadequate immune function commonly results in uncontrolled virus replication, and multi-organ disease and dysfunction. In fact, HCMV significantly contributes to morbidity and mortality in immunocompromised individuals such as newborns, organ transplant recipients, AIDS patients, cancer patients, patients with cardiovascular diseases, autoimmune disease patients, as well as the elderly. HCMV is the leading cause of birth defects affecting 1-4% of newborns in developed countries and significantly contributes to transplant failure. HCMV also increases the severity of cardiovascular diseases, autoimmune diseases, and various cancers. Current therapy against HCMV targets the viral DNA polymerase, but induces severe side effects (e.g. thrombocytopenia) and drug-resistant viral strains upon prolonged treatment. Our central hypothesis is that inhibiting HCMV proliferation by targeting diverse steps of the HCMV life cycle would effectively limit HCMV-associated diseases. Thus, we propose to identify novel therapeutics that inhibit the early steps of HCMV infection utilizing a HCMV variant that expresses a viral protein (immediate-early protein 2) fused to yellow fluorescent protein. A high-content screen will be performed at the NIH Chemical Genomics Core to identify compounds that block the early steps of virus infection. High- throughput secondary assays will be performed to identify lead compounds through their validation in a diverse high-content screen, an effective concentration in the nanomolar range and low toxicity. Also, tertiary assays will define the mode of action of the lead compounds. The discovery of anti-HCMV reagents against the early phase of infection represents a novel class of compounds that will target different proteins than the current therapeutics with the objective o attenuating HCMV proliferation and HCMV-associated diseases in immunocompromised individuals.